Potassium ions are used by the body to build up an electrical potential across cellular membranes. This potential is used as energy to drive the movement of molecules into and out of cells, and, most famously, to drive the electrical activity that characterizes the nervous system. Potassium channels are the proteins that allow these important ions across the membrane, and their structure and function are well known from 60 years of biophysical and structural research. One notable group of potassium channels, known as Slo channels, opens and closes depending on both the membrane voltage and the concentration of various intracellular ions. These channels are involved in diverse elements of physiology, including the timing of neural activity, the function of the inner ear, smooth muscle contraction, and sperm capacitation. Mutations in Slo channels are associated with epilepsy, and they are potential drug targets for a variety of medical purposes. In the Slo family, the best understand channel, Slo1, also called the BK channel, is closely related to the least well understood channel, Slo3. Despite being closely related, the two channel types are affected by different intracellular ions and are expressed in different tissues. Slo1 is sensitive to intracelllar calcium, while Slo3 appears to be primarily sensitive to pH. Slo3 has primarily been characterized in mice, however, and current research suggests human Slo3 might be very different from mice. There are also many outstanding questions about the locus of proton binding in Slo3. My proposed work uses a different approach than the one taken so far in Slo3 research. Rather than look in a few model organisms, I propose to take a comparative and evolutionary approach that can reconstruct the story of how Slo3 came to be different from Slo1. I have used comparative genomics and phylogenetics to learn new things about Slo3 and generate predictive hypotheses about the molecular basis of its function. Contrary to the current belief that Slo3 exists only in mammals, I have found Slo3 genes in the genomes of birds and reptiles. To reconstruct the history of Slo3, I predicted the sequence of the ancestors of extant Slo3 channels using statistical models of evolution. These new channels, extant and ancestral, tell a predictive story of how Slo3 differentiated from Slo1 by losing known calcium binding sites and acquiring new sites that may sense intracellular pH. My proposed work will test these hypotheses using heterologous expression and biophysics. The work will be done in the laboratory of Dr. Richard Aldrich, who has contributed much of the current knowledge of Slo channel function.